The present invention, in some embodiments thereof, relates to a 4′-O-glycosyltransferase and, more particularly, but not exclusively, to the use of same for producing trilobatin and for engineering cells and plants with increased trilobatin content.
Apple (Malus×domestica Borkh.) is an agriculturally and economically important source of food and beverage. Apples accumulate high levels of diverse phenolic antioxidants, including flavonoids and biphenyl phytoalexins, the level of which may depend on many factors, such as genotype, developmental stage, or biotic stress.
Among common phenylpropanoid derivatives, apples are characterized by the presence of dihydrochalcones (DHCs), structures closely related to common flavonoid precursors, the chalcones. Like other flavonoids, DHCs exhibit a wide diversity of hydroxyl and glucosyl substitution patterns. 3-OH-phloretin, phloridzin (phloretin-2′-O-glucoside) (illustrated by no. 7 in FIG. 4), trilobatin (phloretin-4′-O-glucoside) (illustrated by no. 6 in FIGS. 1 and 4) and sieboldin (3-hydroxyphloretin-4′-O-glucoside) accumulate in different combinations in the stems, leaves, flowers and fruits of apple plants.
The physiological function of the DHCs planta remains unresolved. They have been suggested to act as UV filters in leaves or to play a role as potent antioxidants and in resistance to pathogens, but conclusive evidence is still missing. For instance, phloretin (illustrated by no. 5 in FIGS. 1 and 4), a simple dihydrochalcone found in the plant kingdom, has inhibitory activity against the glucose co-transporter 1 and a proven antioxidant activity. DHCs have been reported to display anti-carcinogenic activity, inhibit cardiovascular diseases or to be a potential drug for Alzheimer's disease. Despite their high potential as drugs and food additives the use of DHCs is still limited due to water insolubility and low bioavailability, which in part can be improved by glycosylation.
Glucosylation at the 4′-position of phloretin (illustrated by no. 5 in FIGS. 1 and 4) generates a sweet tasting compound, trilobatin (illustrated by no. 6 in FIGS. 1 and 4), also known as pruning dihydrochalcone. Trilobatin has been previously detected in the leaves of Vitis species, in Lithocarpus polystachyus, and is also present in different Malus species including M. domestica and M. trilobata. Other dihydrochalcone derivatives are sweet tasting as well; neohesperidin dihydrochalcone (NHDC) and naringin dihydrochalcone, the latter found to accumulate in Oxytropis myriophylla, are up to 1000 fold sweeter than sucrose per weight unit, and are stable under heat and a wide pH range. Dihydrochalcones have also been documented to function as bitterness blockers and flavor enhancers with diverse use in the food, beverage and pharmaceutical industries. Altogether, about 200 different dihydrochalcones are known from 30 plant families, including Camellia japonica, Fragaria×ananassa, Lithocarpus polystachyus, legumes and Citrus species.
The biosynthetic pathway leading to trilobatin in planta has not been fully resolved as yet. The condensation of one molecule of 4-dihydrocoumaroyl-CoA (illustrated by no. 3 in FIG. 1) and three molecules of malonyl-CoA is catalyzed by chalcone synthase to yield phloretin (as illustrated in FIG. 1).
The attachment of a glucose moiety to phloretin at the 4′ position is suggested to be the final step in the formation of trilobatin. Ono et al. [Ono E. et al., P. Natl. Acad. Sci. USA (2006) 103: 11075-11080] have shown that glycosylation of chalcones can be catalyzed by a recombinant chalcone 4′-O-glucosyltransferase isolated from snapdragon (Antirrhinum majus). Recently, a functionally expressed UDP-glucosyltransferase (UGT) from a bacteria, Bacillus lichentfomis, has been shown to catalyze the conversion of phloretin to trilobatin [Pandey R. P. et al., Appl. Environ. Microb. (2013) 79, 3516-3521].
U.S. Patent Application No. 20110030098 discloses a method for producing a plant cell or plant with increased phlorizin or phloretin glycosyltransferase activity by transformation of the plant cell or plant with a polynucleotide encoding a polypeptide with phloretin glycosyltransferase activity. U.S. 20110030098 also provides host cells, plant cells and plants, genetically modified to contain and or express the polynucleotides.